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782. Transduction of Primary Human Monocyte-Derived Macrophages and Dendritic Cells by Recombinant SV40 Vectors
GENE THERAPIES AND VACCINES AGAINST INFECTIOUS DISEASES 782. Transduction of Primary Human MonocyteDerived Macrophages and Dendritic Cells by Recombinant SV40 Vectors Sandra A. Calarota,1 Miguel Otero,1 Pierre Cordelier,2 Roger J. Pomerantz,1 David S. Strayer.2 1 Division of Infectious Diseases, Department of Medicine, Thomas Jefferson University, Philadelphia, PA, United States; 2 Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA, United States. Macrophages and dendritic cells (DC) are important potential targets for the genetic therapy of infectious diseases and cancer. Gene transfer to macrophages and DC has been difficult to achieve using conventional vector systems because these cells are terminally differentiated and nondividing, and their natural phagocytic functions lead to degradation of the phagocytosed vector particles. Recombinant SV40-derived vectors (rSV40s) transduce resting cells efficiently. Also, they enter cells via caveolae, bypassing the phagocytic process. We tested whether rSV40s could transduce macrophages and DC effectively and, in the process, deliver transgene constructs designed to protect these cells from infection with HIV-1. For this purpose, we used two different rSV40s: SV(nef-FLAG), in which the HIV-1 protein Nef carrying a carboxyl terminal FLAG epitope tag is driven by the CMV promoter; and SV[HIVLTR]IFN, in which expression of the cDNA for human interferon alpha-2 (IFNα2) is driven by the HIV-1 long terminal repeat (LTR) as a promoter. DC and primary human monocyte-derived macrophages (MDM) were prepared from buffy coat peripheral blood monocytes. Monocytes were isolated by magnetic sorting for CD11b. These cells were cultured with GM-CSF and M-CSF for 2 weeks to produce MDM. DC were prepared by sorting for CD14, and were induced to mature using TNF-α. Terminally differentiated MDM were treated with SV[HIVLTR]IFN or a control rSV40, SV(HBS), which carries the hepatitis B surface antigen. Transduced MDM were challenged with HIV-1BaL, or mock-challenged. Expression of IFNα, induced by HIV-1 infection, was assessed by immunostaining. Macrophages express a low level of IFNα naturally. Challenge with HIV-1 greatly increased the level of IFNα production in SV[HIVLTR]IFN-transduced cells. Controltransduced and control-challenged cells revealed very low levels of IFNα by immunostaining. We also tested whether SV[HIVLTR]IFN could deliver sufficient IFNα to inhibit HIV-1 replication in MDM. Following challenge with HIV-1BaL, supernatant HIV-1 p24 antigen concentrations were measured as a function of time by ELISA. Significant and substantial inhibition of HIV-1 replication was observed in SV[HIVLTR]IFN-transduced MDM, compared with control-transduced MDM. In parallel, we tested the ability of rSV40 vectors to transduce primary DC. SV(nef-FLAG), encoding an HIV-1 Nef protein with a carboxyl terminal FLAG epitope tag, was used for this purpose. Nef-FLAG expression was detected by immunostaining for the FLAG epitope. By immunostaining, we demonstrated that SV(nefFLAG) transduced both immature and mature dendritic cells quite efficiently. Therefore, we demonstrated that primary human macrophages and dendritic cells can be efficiently transduced with recombinant SV40based vectors. Further, delivery of a conditional expression construct, in which expression of human IFNα is activated by HIV-1 infection, can inhibit HIV-1 replication in these cell-types.
783. Combination Gene Therapy Using rSV40 as a Vehicle: Inhibiting CCR5-Dependent HIV-1 Infection of Primary Macrophages with a Hammerhead Ribozyme and a Single Chain Antibody Pierre Cordelier,1 Laurence Cagnon,2 John Rossi,2 David S. Strayer.1 1 Pathology, Jefferson medical college, Philadelphia, PA, United States; 2City of Hope, Beckman Institute, Duarte, CA, United States. Background: the CCR5 chemokine receptor is important in HIV entry of monocytes and fixed phagocytes. These cells are usually the first targets when HIV enters the body. Studies of naturally occurring polymorphisms of the CCR5 gene have shown that its deletion or reduced expression has no detectable negative consequences and can impede HIV1 infection. Since CCR5 appears to be dispensable for the host, but important for initial HIV-1 infection, CCR5 is an excellent target for elimination by gene therapy. Recombinant SV40 vectors (rSV40) were
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tested for their ability to deliver a CCR5-specific single-chain antibody and a hammerhead ribozyme against CCR5 in primary monocyte-derived macrophages (MDM) and to inhibit HIV-1 replication. Methods: the rSV40 vectors used in these studies carried a CCR5-specific single-chain antibody driven by CMV immediate early promoter (SV(2C7)) and a hammerhead ribozyme against CCR5 driven by an adenovirus pol III promoter (SV(VAKA1)). Monocytes are purified from normal peripheral blood monocytes from normal volunteers by selective adherence to plastic, cell sorting and then maintained in culture with GM-CSF and C-CSF to allow differentiation into MDM for 2 weeks. MDM were transduced with SV(2C7) and/or SV(VAKA1). No selection was used. CCR5 expression was assayed by Northern blot and immunostaining. MDM were challenged with HIV1 BaL. Subsequent HIV-1 replication was measured by ELISA as supernatant p24 antigen levels. Results: SV(2C7) and SV(VAKA1) transduced primary MDM without detectable toxicity. By direct immunostaining, >90% of unselected cells were transduced by SV(2C7). The hammerhead ribozyme against CCR5 efficiently reduced cellular CCR5 mRNA when stably introduced into MDM by transduction with SV(VAKA1). These two transgenes both blocked surface expression of human CCR5, when analyzed using immunostaining. MDM treated with SV(2C7) and SV(VAKA1) strongly resisted challenge with 0.1, 0.5 and 1 ng of HIV-1 BaL. Protection by these transgenes individually was overcome by 1.5 ng HIV-1 BaL. But was restored by combined transduction of MDM with SV(2C7) and SV(VAKA1). Conclusions: Reducing the concentration of CCR5 receptor at the cell membrane using SV(2C7) and SV(VAKA1) produced human primary monocyte-derived macrophages resistant to HIV-1 infection. These findings suggest a novel gene therapeutic approach to HIV treatment using rSV40 as a delivery vehicle. A very high challenge doses of HIV-1 BaL could overwhelm the protective effect of single transgenes, but was inhibited by co-transduction of both together. Therapeutic combinations provided optimum protection to human MDM transduced with SV(2C7) and SV(VAKA1). Use of rSV40 vectors readily lends itself to multiple therapeutic combinations or multiple administrations to provide optimum protection where and when very high HIV-1 concentrations may be attained.
α2 Delivered to T Cells by a 784. Trans-Activated IFN-α Recombinant SV40 Vectors Inhibits Early Stages in the HIV-1 Replicative Cycle Pierre Cordelier,1 Sandra A. Calarota,1 David S. Strayer.1 1 Pathology, Jefferson Medical College, Philadelphia, PA, United States. Background: several lines of evidence suggest a potential role for IFN alpha (IFN-α) in control of HIV-1 replication. However, recombinant IFN-α protein is only moderately effective in inhibiting HIV, and such inhibition is reversible when the IFN therapy is withdrawn. We hypothesized that stable expression of human IFN-α2, delivered by recombinant SV40 vectors (rSV40), and conditional on HIV infection, could provide sustained protection from HIV. Methods: to achieve high levels of expression of IFN-α2 at the site of infection, we devised a recombinant, Tag-deleted, SV40-derived gene transfer vector, SV[HIVLTR]IFN, in which expression of human IFN-α2 would be driven by the HIV-1 LTR and so be trans-activated by HIV infection. Human T lymphocyte cell lines and primary human blood lymphocytes (PBL) were transduced with SV[HIVLTR]IFN. No selection was used. There was no detectable toxicity. These and control cultures were challenged with HIV-1NL4-3. HIV-1 replication was measured by ELISA as supernatant p24 antigen levels. Results: Expression of IFN-α2 was strongly induced by HIV-1 challenge, as indicated by Northern and Western blotting in SV[HIVLTR]IFN-transduced, HIV-challenged human lymphocytic cell lines and PBL. SV[HIVLTR]IFN-transduced cells were totally protected from HIV-1 related cytophatic effects. As measured by HIV-1 p24 antigen assays, IFN-α2 expressing cell lines and PBL were also protected from HIV-1 replication. We tested the mechanism of IFN-α2 inhibition of HIV-1, and found that rSV40-delivered IFN-α2 was associated with inhibition of HIV1 protein production, as measured by Western analysis for gp120, and decreased expression of HIV mRNAs as shown by RT-PCR. Finally, Southern analysis revealed that levels of unintegrated proviral DNA were markedly reduced in SV[HIVLTR]IFN-transduced cells compared to control cultures. Conclusions: Delivering IFN-α2 expression, driven by HIV-1 LTR, to human lymphocyte cell lines or PBL using SV[HIVLTR]IFN Molecular Therapy Vol. 5, No. 5, May 2002, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
783. Combination Gene Therapy Using rSV40 as a Vehicle: Inhibiting CCR5-Dependent HIV-1 Infection of Primary Macrophages with a Hammerhead Ribozyme and a Single Chain Antibody Pierre Cordelier,1 Laurence Cagnon,2 John Rossi,2 David S. Strayer.1 1 Pathology, Jefferson medical college, Philadelphia, PA, United States; 2City of Hope, Beckman Institute, Duarte, CA, United States. Background: the CCR5 chemokine receptor is important in HIV entry of monocytes and fixed phagocytes. These cells are usually the first targets when HIV enters the body. Studies of naturally occurring polymorphisms of the CCR5 gene have shown that its deletion or reduced expression has no detectable negative consequences and can impede HIV1 infection. Since CCR5 appears to be dispensable for the host, but important for initial HIV-1 infection, CCR5 is an excellent target for elimination by gene therapy. Recombinant SV40 vectors (rSV40) were
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tested for their ability to deliver a CCR5-specific single-chain antibody and a hammerhead ribozyme against CCR5 in primary monocyte-derived macrophages (MDM) and to inhibit HIV-1 replication. Methods: the rSV40 vectors used in these studies carried a CCR5-specific single-chain antibody driven by CMV immediate early promoter (SV(2C7)) and a hammerhead ribozyme against CCR5 driven by an adenovirus pol III promoter (SV(VAKA1)). Monocytes are purified from normal peripheral blood monocytes from normal volunteers by selective adherence to plastic, cell sorting and then maintained in culture with GM-CSF and C-CSF to allow differentiation into MDM for 2 weeks. MDM were transduced with SV(2C7) and/or SV(VAKA1). No selection was used. CCR5 expression was assayed by Northern blot and immunostaining. MDM were challenged with HIV1 BaL. Subsequent HIV-1 replication was measured by ELISA as supernatant p24 antigen levels. Results: SV(2C7) and SV(VAKA1) transduced primary MDM without detectable toxicity. By direct immunostaining, >90% of unselected cells were transduced by SV(2C7). The hammerhead ribozyme against CCR5 efficiently reduced cellular CCR5 mRNA when stably introduced into MDM by transduction with SV(VAKA1). These two transgenes both blocked surface expression of human CCR5, when analyzed using immunostaining. MDM treated with SV(2C7) and SV(VAKA1) strongly resisted challenge with 0.1, 0.5 and 1 ng of HIV-1 BaL. Protection by these transgenes individually was overcome by 1.5 ng HIV-1 BaL. But was restored by combined transduction of MDM with SV(2C7) and SV(VAKA1). Conclusions: Reducing the concentration of CCR5 receptor at the cell membrane using SV(2C7) and SV(VAKA1) produced human primary monocyte-derived macrophages resistant to HIV-1 infection. These findings suggest a novel gene therapeutic approach to HIV treatment using rSV40 as a delivery vehicle. A very high challenge doses of HIV-1 BaL could overwhelm the protective effect of single transgenes, but was inhibited by co-transduction of both together. Therapeutic combinations provided optimum protection to human MDM transduced with SV(2C7) and SV(VAKA1). Use of rSV40 vectors readily lends itself to multiple therapeutic combinations or multiple administrations to provide optimum protection where and when very high HIV-1 concentrations may be attained.
α2 Delivered to T Cells by a 784. Trans-Activated IFN-α Recombinant SV40 Vectors Inhibits Early Stages in the HIV-1 Replicative Cycle Pierre Cordelier,1 Sandra A. Calarota,1 David S. Strayer.1 1 Pathology, Jefferson Medical College, Philadelphia, PA, United States. Background: several lines of evidence suggest a potential role for IFN alpha (IFN-α) in control of HIV-1 replication. However, recombinant IFN-α protein is only moderately effective in inhibiting HIV, and such inhibition is reversible when the IFN therapy is withdrawn. We hypothesized that stable expression of human IFN-α2, delivered by recombinant SV40 vectors (rSV40), and conditional on HIV infection, could provide sustained protection from HIV. Methods: to achieve high levels of expression of IFN-α2 at the site of infection, we devised a recombinant, Tag-deleted, SV40-derived gene transfer vector, SV[HIVLTR]IFN, in which expression of human IFN-α2 would be driven by the HIV-1 LTR and so be trans-activated by HIV infection. Human T lymphocyte cell lines and primary human blood lymphocytes (PBL) were transduced with SV[HIVLTR]IFN. No selection was used. There was no detectable toxicity. These and control cultures were challenged with HIV-1NL4-3. HIV-1 replication was measured by ELISA as supernatant p24 antigen levels. Results: Expression of IFN-α2 was strongly induced by HIV-1 challenge, as indicated by Northern and Western blotting in SV[HIVLTR]IFN-transduced, HIV-challenged human lymphocytic cell lines and PBL. SV[HIVLTR]IFN-transduced cells were totally protected from HIV-1 related cytophatic effects. As measured by HIV-1 p24 antigen assays, IFN-α2 expressing cell lines and PBL were also protected from HIV-1 replication. We tested the mechanism of IFN-α2 inhibition of HIV-1, and found that rSV40-delivered IFN-α2 was associated with inhibition of HIV1 protein production, as measured by Western analysis for gp120, and decreased expression of HIV mRNAs as shown by RT-PCR. Finally, Southern analysis revealed that levels of unintegrated proviral DNA were markedly reduced in SV[HIVLTR]IFN-transduced cells compared to control cultures. Conclusions: Delivering IFN-α2 expression, driven by HIV-1 LTR, to human lymphocyte cell lines or PBL using SV[HIVLTR]IFN Molecular Therapy Vol. 5, No. 5, May 2002, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy